Ion exchangers have been used for many years to separate out proteins from protein-containing solutions. They have found application in the dairy industry, particularly in recovering whey proteins from milk and milk derived process streams, such as whey and whey protein concentrates.
Ion exchangers which have been used in separating whey proteins from whey protein containing solutions include both cation exchangers, particularly of the SP or SE (sulphonate) or CM (carboxymethyl) type, and anion exchangers, particularly of the QA (quaternary amino) or DEAE (diethylaminoethyl) type. In terms of the exchanger matrix itself, many insoluble matrices have been used, including cellulose, cross-linked dextran, cross-linked agarose, synthetic hydophilic polymers and inorganic materials coated with hydrophilic polymers.
One matrix that has proved to be particularly useful in large scale separation and purification of whey proteins is regenerated cellulose which has been hydroxyalkylated and cross-linked. Ion exchangers prepared on this matrix are resistant to attrition, have high protein capacity, high flow properties and are available at relatively low cost.
Examples of such ion exchangers based on a hydroxyalkylated and cross-linked regenerated cellulose matrix which are commercially available include the SP, CM, QA, and DEAE exchangers sold as SP GibcoCel™, CM GibcoCel™, QA GibcoCel™ and DEAE GibcoCel™ respectively. These ion exchangers were previously sold under the Indion™ brand name. QA GibcoCel™ and SP GibcoCel™ having a substitution level of the QA or SP groups of up to 1.2 milli-equivalents per dry gram (meq/g) are available. SP GibcoCel™, a cation exchanger, has been widely used, but QA GibcoCel™, an anion exchanger, has only enjoyed limited use industrially.
Levison et al (Chimica Oggi/Chemistry Today, 41-48, November/December 1994) refers to three custom made QA celluloses with substitution levels of 0.74, 0.96 and 1.24 meq/g, and discloses that these had similar protein capacities.
Anion exchangers bearing quaternary amino (QA) groups are typically made by alkylation of either a hydrophilic hydroxyl-bearing matrix or such a matrix already bearing tertiary amino groups such as diethylaminoethyl (DEAE) groups. In the latter case simple alkylating agents may be used such as ethylene oxide as shown in the following equation. 
Direct alkylation of the hydrophilic matrix is achieved using agents already containing a quaternary ammonium group, eg 
Several such reagents are summarized in U.S. Pat. No. 5,731,259 and many of them are available commercially for large scale industrial use.
Alkylating agents (3-chloro-2-hydroxypropyl)trimethylammonium chloride (CHPTAC) and glycidyltrimethylammonium chloride (GTAC) have been widely used to prepare quaternary ammonium derivatives (cationic derivatives) of polysaccharides, especially starch and cellulose. Both water soluble and water insoluble derivatives have been prepared for a variety of purposes. Only the latter are useful as anion exchangers for the adsorption and chromatography of proteins.
Japanese patent 79042385 (1979) and Chemical Abstracts 91, 58084 (Toyo Pulp KK) describe the preparation of a crosslinked QA cellulose with a degree of substitution (DS) of 0.13 (<<1 meq/g) and a protein capacity of 0.24 g/g, using 50% CHPTAC.
CS 202,374 (1983) and Chemical Abstracts 99, 72429 describe the preparation of ion exchangers with capacities of 0.37 to 0.68 meq/g from powdered cellulose. Analogous products were also obtained from crosslinked cellulose, hydroxyethyl cellulose and starch and stated to be useful as ion exchangers, sorbents and flocculants.
A further Czechoslovakian patent, CS 236,024 (1987), and Chemical Abstracts 109, 151774 describe the preparation of trimethylammoniumhydroxypropyl cellulose, an ion exchanger with an exchange capacity of 0.24 meq/g after first activating the cellulose with acetic or phosphoric acid.
Several 1989 Japanese patents, JP 01/130,726; 01/106,898 and 01/099,646 (Daicel Chemical Industries, Ltd) (Chemical Abstracts 112, 95049; 112, 135584 and 113, 20528) disclose the preparation of crosslinked, cationized hydroxy-alkylcellulose gels for chromatography of nucleic acids. For example, hydroxyethyl cellulose is reacted with GTAC or CHPTAC and crosslinked and used to bind nucleic acids selectively from a mixture of nucleic acids and proteins. Low substitution levels of QA groups are typically useful for binding nucleic acids but not proteins, hence the selectivity observed.
WO 91/17830 describes the use of regenerated cellulose to prepare a crosslinked flexible sponge with fibrous reinforcement. This was then derivatized by reaction with CHPTAC to give a QA cellulose sponge with a protein binding capacity of 1.5 g/g. Such products have yet to be made and demonstrated on the very large scale needed for use in the dairy industry.
Antal et. al. (Carbohydrate Polymers 19, 167-169, 1992) describe the optimization of the reaction of microcrystalline cellulose with the alkylating agents CHPTAC and 1,3-bis(3-chloro-2-hydroxy-propyl)imidazolium hydrogen sulfate in alkaline medium. The maximum substitution level they were able to obtain with CHPTAC was 0.94 meq/g (mmol/g), although the second reagent gave a product with 1.56 meq/g. No protein capacities are given and it is likely that the latter reagent, being bifunctional, would have introduced extensive crosslinking into the cellulose to the detriment of protein capacity. Furthermore microcrystalline cellulose is not a suitable matrix for repeated use on a large industrial scale.
CHPTAC has been used to make a bead-shaped QA starch anion exchanger with exchange capacity of 0.90 meq/g. (Chemical Abstracts 130, 63153, 1998). The corresponding diethylaminoethyl (DEAE) starch made using 2-chloroethyl(diethyl)amine hydrochloride had a capacity of 2.47 meq/g showing the greater difficulty typically experienced in making the quaternary amino (QA) derivatives than for the tertiary amino derivatives like DEAE.
Fibrous cellulose has been derivatized with quaternary ammonium groups to a high degree of substitution, DS of at least 0.5 (>2 meq/g), using a very large excess of alkylating reagent containing quaternary ammonium groups. The cellulose is either not crosslinked (1998 U.S. Pat. No. 5,731,259) or crosslinked (1998 U.S. Pat. No. 5,780,616). Preferably the alkylating reagent is used in 20:1 to 40:1 mole ratio of reagent to anhydroglucose units of cellulose. In the case of GTAC this amounts to 186-372 g of reagent per 10 g of cellulose used either in 5-8 repeated reactions or one large addition of the solid reagent with 30 mL of water. The products, described at one point as a jelly mass, are useful as superabsorbents for water and saline solutions in the field of hygenic-sanitary products such as diapers for babies. They are designed to be used once and then disposed of and are not at all suitable for repeated use day after day in a reactor or column bed where physical robustness against attrition, long life and high flow-through rates are required for anion exchangers processing protein solutions.
With the above background in mind, it was an object of the present invention to provide an anion exchanger which is particularly useful on an industrial scale in separating whey proteins from whey protein containing solutions, or at least to provide the public with a useful choice.